Use of a polysaccharide excreted by the Vibrio diabolicus species in bone repair

ABSTRACT

The invention concerns the use of a polysaccharide excreted by the  Vibrio diabolicus  species for making a medicine with cicatrising activity, in particular bone repair activity. The invention also concerns a bone reconstruction biomaterial comprising said polysaccharide and a bone endoprosthesis whereof the surface is coated with such a polysaccharide.

The present invention relates to the use of a polysaccharide excreted bythe Vibrio diabolicus species for producing a medicinal product withcicatrizing activity, and also to a bone reconstruction biomaterialcomprising this polysaccharide.

Bone substance losses are disabling and are slow to repair. When thebone loss exceeds a critical size, no spontaneous repair can occur (J.P. Schmitz et al., Acta Anat., 1990, 138, 185–192). Bone-fillingmaterials, which conduct or induce repair, must then be used in order torestore continuity between the two sides of the bone loss.

The most long-standing transplantation techniques consist in usingcomplete bone, which inherently comprises proteins capable of initiatingand promoting the biological mechanisms of bone reconstruction, such asBMPs or bone morphogenetic proteins, osteocalcin, osteopontin andosteogenin. Autografts have the advantage of being completely toleratedby the immune system. However, the bone store is limited in anindividual and the additional surgical intervention which ensuestherefrom leads to risks of complications. As regards allografts orxenografts, they present risks of pathogenic agent transmission and maylead to rejection phenomena.

Due to the inadequacies of bone grafts, research has been directedtoward bone substitute materials. These filling materials may beosteoinductive or osteoconductive materials.

Osteoinductive materials enable regeneration of the tissue lossaccording to a mechanism of cell activation by the proteins or peptidesof the filler material. They are capable of inducing ossification at anectopic site, i.e. extra-osseous ossification. By way of examples ofosteoinductive substances and materials other than bone in its naturalform, mention may be made of:

-   -   growth factors and hormones (in particular cytokines, IGFs or        insulin-like growth factors, and FGFs or fibroblast growth        factors) for which it has been demonstrated that they promote        bone reconstruction (P. A. Hill et al., Endocrinology, 1995,        136(1), 124–131: P. Cuevas et al., Surg. Neurol., 1997, 47,        242–246; G. L. Barnes et al., Journal of Bone and Mineral        Research, 1999, 14(11), 1805–1815). M. Isobe et al. (Journal of        Biomedical Materials Research, 1996, 32, 433–438) have in        particular proposed injecting rats subcutaneously with growth        factors of the BMP type, extracted from bone tissue. BMPs, and        also, in general, the other growth factors and hormones, give        random results, however, when they are used clinically. The        systemic effect thereof, i.e. effect not limited to the site of        implantation, presents risks of diffusion of BMP into the        neighboring tissues of the damaged site, and of local        calcification at a non-osseous site. In addition, the use of        BMPs may lead to inter- or intraspecies contaminations;    -   dextran derivatives bearing functional groups chosen from        carboxymethyl, benzylamide, sulfate and sulfonate (called        “CMDBS”) and heparan sulfates, which bind the growth factors        present at the lesion, protecting them from enzymatic        degradation (D. Aviezer et al., The Journal of Biological        Chemistry, 1994, 269(1), 114–121; F. Blanquaert et al., Journal        of Biomedical Materials Research, 1999, 44, 63–72; M. L.        Colombier et al., Journal de Parodontologie et d'Implantologie        Orale, 1998, 17(4), 403–413; M. Tardieu et al., Journal of        Cellular Physiology, 1992, 150, 194–203). These compounds        nevertheless require a vector, such as collagen, in order to be        used (A. Meddahi et al., Diabetes & Metabolism, 1996, 22,        274–278; A. Meddahi et al., Path. Res. Pract., 1994, 190,        923–928). A. Meddahi et al. (1994, ibid) have demonstrated that        a particular dextran derivative, called CMDBS K (comprising 83%        of carboxymethyl groups, 23% of benzylamide groups and 13% of        sulfonate groups), soaked in a collagen buffer, makes it        possible to partially fill, after three weeks, bone defects 3 mm        in diameter produced in rat calvaria. These results cannot,        however, be extrapolated to critical size lesions, the reference        critical diameter for such a period of time being 5 to 8 mm.

Moreover, osteoconductive materials, as their name indicates, merelyconduct the repair, without inducing any bone formation per se: theyserve as a support for the biological phenomena of bone repair and areintended to be resorbed after a certain amount of time, and replacedwith the newly formed bone tissue. By way of examples of osteoconductivematerials, mention may be made of:

-   -   bone in all its deproteinized and/or ceramized forms, of human        or bovine origin for example (F. A. Papay et al., The Journal of        Craniofacial Surgery, 1996, 7(5), 347–351);    -   synthetic or natural calcium phosphates, such as tricalcium        phosphate, biphasic calcium phosphate, hydroxyapatite, or corals        (F. A. Papay et al., ibid; N. B.-A. Naaman et al., The        International Journal of Oral and Maxillofacial Implants, 1998,        13(1), 115–120; M. Trecant et al., Clinical Materials, 1994, 15,        233–240);    -   biovitroceramics, bioglasses.

In view of the inadequacies and the disadvantages of this state of theart in terms of bone reconstruction, the inventors therefore gavethemselves the aim of providing a biomaterial which can be used in bonereconstruction, and which is suitable for the reconstruction of largelosses of bone substances, i.e. of defects the size of which is greaterthan the critical size. This biomaterial must be biocompatible,nonimmunogenic and resorbable at the end of bone reconstruction.

Surprisingly, the inventors have discovered that these aims are achievedusing a particular polysaccharide, namely a polysaccharide excreted bythe Vibrio diabolicus species.

A subject of the invention is the use of a polysaccharide excreted(termed “exopolysaccharide”) by the Vibrio diabolicus species whosestrain was deposited with the CNCM (Collection nationale de Cultures deMicroorganismes [National collection of cultures of microorganisms], 28rue du Docteur Roux; 75724 Paris, France) on Oct. 17, 1995, under thenumber I-1629, for producing a medicinal product with cicatrizingactivity.

The physicochemical characteristics and the metabolite properties of theVibrio diabolicus species (isolated from the strain HE 800, belonging tothe Vibrio genus) are described in the PCT International application inthe name of IFREMER, published under the number WO 98/38327.

The medicinal product with cicatrizing activity defined above inparticular has bone repair activity, for example with a view topreparing a bone repair or filling material, such as bone endoprostheses(dental prostheses and joint prostheses for example).

Particularly advantageously, the polysaccharide defined above may beused to prepare a coating for a bone endoprosthesis, or for anosteoconductive filling material. Osteoconductive filling materials orbone endoprostheses (such as dental bone endoprostheses made of titaniumor joint prostheses made of titanium without orthopedic cement) coatedwith such a polysaccharide can in fact integrate very rapidly into therecipient bone.

A subject of the invention is also a bone endoprosthesis, characterizedin that at least part of its surface is coated with the polysaccharideas defined above.

A polysaccharide which can be used in the invention is, for example, apolysaccharide which can be obtained by precipitation with ethanol fromculture supernatants of said Vibrio diabolicus species. Thispolysaccharide is such that it:

-   -   does not comprise any neutral polysaccharides,    -   has an acid saccharide content of approximately 50% by weight,    -   has an osamine content of approximately 50% by weight,    -   has a glucuronic-acid/N-acetylgalactosamine/N-acetylglucosamine        monosaccharide molar ratio of approximately 2/1/1.

The structure of such a polysaccharide is explained in detail in thearticle by H. Rougeaux et al., published in Carbohydrate Research, 1999,322, 40–45. It may be used in its native form or in a derived chemicalform, the polysaccharide being, for example, functionalized with sulfategroups.

The polysaccharide used in the invention may be in a dry form, i.e. in acottony, fibrous or pulverulent form, depending on the final treatmentto which it is subjected (grinding makes it possible to obtain a powder,lyophilization results in a polymer with a cottony appearance, anddrying makes it possible to obtain fibers). The polysaccharide used inthe invention may also be in a hydrated form, for example in the form ofa hydrogel. It might also be possible to envisage using such apolysaccharide in the form of a membrane or of a solid three-dimensionalalveolate structure.

As a variant, the polysaccharide used in the invention may be combinedwith an osteoconductive and/or osteoinductive material.

Such an osteoconductive material may in particular be selected from thegroup consisting of deproteinized and/or ceramized bone, such as thebones marketed respectively by the laboratory S.P.A.D. (Quetigny,France) and the company OSTE (Clermont-Ferrand, France) under the namesLaddec® and Lubboc®, synthetic or natural calcium phosphates (such asβ-form tricalcium phosphate, biphasic calcium phosphate, hydroxyapatiteor corals), biovitroceramics and bioglasses (for example Bioglas® andPerioglas® marketed by the laboratory PHARMADENT (Levallois-Perret,France).

As regards the osteoinductive material which can be combined with thepolysaccharide used in the invention, it may be a hormone or a growthfactor, in particular BMPs. Combining said polysaccharide with anosteoinductive material makes it possible to increase the rate ofrepair, for example by supplying BMPs directly to the damaged site. Thepolysaccharide used in the invention, excreted by the Vibrio diabolicusspecies, serves as a support for the osteoinductive material andprotects it from enzymatic degradation.

A subject of the invention is also a bone reconstruction biomaterial,characterized in that it comprises a polysaccharide excreted by theVibrio diabolicus species whose the strain was deposited with the CNCMon Oct. 17, 1995, under the number I-1629, and also an osteoconductiveand/or osteoinductive material, for example such as those describedabove.

Besides the above arrangements, the invention also comprises otherarrangements which will emerge from the following description, whichmakes reference to examples of use of the polysaccharide produced by theVibrio diabolicus bacterium in bone repair. It should be clearlyunderstood, however, that these examples are given only by way ofillustration of the subject of the invention, of which they in no wayconstitute a limitation.

EXAMPLE 1 Obtaining an Exopolysaccharide Produced by the VibrioDiabolicus Bacterium

-   a) Vibrio diabolicus cultures

The strain HE 800 is cultured on 2216E medium (Oppenheimer, J. Mar.Res., 1952, 11, 10–18) enriched with glucose (40 g/l). The production iscarried out at 30° C. and at pH 7.4 in a 2 liter fermenter containing 1liter of the glucose-containing 2216E medium, as described by P. Vincentet al. in Appl. Environ. Microbiol., 1994, 60, 4134–4141. Afterculturing for approximately 48 hours, the culture medium has a viscosityof the order of 100 centipoises at 60 rpm.

-   b) Purification of the exopolysaccharide

After 50/50 dilution with distilled water, the bacteria are separatedfrom the culture medium by centrifugation at 20 000 g for 2 hours.Sodium chloride is then added to the diluted solution in such as way asto achieve a concentration of this salt of 20 g/l. The solution ismaintained at ambient temperature and the polysaccharide is precipitatedfrom the supernatant using pure ethanol at 4° C. The polysaccharide isrecovered and is then subjected to several washes with ethanol/waterwith increasing proportions of ethanol (70/30, 80/20, 90/10 and 100/0 byvolume), in accordance with the method described by F. Talmont et al.(Food Hydrocolloids, 1991, 5, 171–172) or by P. Vincent et al. (ibid).

The polymer obtained is dried at 30° C. and conserved at ambienttemperature. Approximately 4.5 g of purified polysaccharide per liter ofculture were thus obtained.

EXAMPLE 2 Use of the Exopolysaccharide Produced by the Vibrio DiabolicusBacterium, in vivo, as Bone Filling Biomaterial

-   a) Protocol

10 6- to 7-week-old male Wistar rats weighing 275 to 299 g are used. Therats are subjected to parenteral anesthesia using Nesdonal® (0.1 ml/100g, Specia Rhône Poulenc Rorer, Montrouge, France), after intramuscularsedation with Ketamine (100 mg/kg) in the Imalgene® formulation (Mérial,Lyons, France) and subcutaneous premedication with Robinul® (0.01 mg/kg,Vetoquinol, Lure, France) half an hour before anesthesia.

Burr holes 5 mm in diameter are made in the calvaria of the rats, inboth parietal lobes, on either side of the mid-sagittal suture,according to the protocol described by P. Cuevas et al. (Surg. Neurol.,1997, 47, 242–6) or by C. Bosch et al. (Cleft Palate CranofacialJournal, 1995, 32(4), 311–317): the animals' cranium is shaved usingclippers and then a two-blade razor. Pre- and postoperative disinfectionof the surgical site is performed with 10% dermal Betadine (Asta Medica,Merignac, France). A median incision is made over approximately 20 mmand the cutaneous tissues are bent back, as is the periosteum. The burrholes, under irrigation with physiological saline, are made withball-shaped diamond cutters, reference 801.104.014, 1.4 mm in diameter(Komet, France). An orifice is made on the right parietal, and anotheron the left parietal, taking care not to damage the dura mater.

Once the two burr holes have been made, the exopolysaccharide obtainedin example 1 (between 1 and 2 mg) is positioned, in its native form (drystructure with an appearance similar to cotton), in the cavity made inthe right parietal, whereas the second cavity is not filled and will beused as a control. The periosteum is sutured using a Vicryl® 3.0resorbable thread (Johnson & Johnson Intl., Brussels, Belgium) and thesutures for the cutaneous layers are made of a nonresorbable material.

The animals are then placed in cages, and sacrificed by injection ofpentobarbital (Doléthal®, Vétoquinol, Lure, France) after two weeks. Thecalvaria are removed, fixed with formaldehyde and demineralized beforebeing studied histologically.

-   b) Results

The polysaccharide excreted by the Vibrio diabolicus bacterium enablesbone repair by filling, for all the animals tested, the cavities made inthe calvaria of the rats. From the histological point of view, noinflammatory reactions are noted, the polysaccharide used is no longerdetectable and the bone newly formed in two weeks is perfectlystructured: the collagen fibers are oriented, the osteoblasts cover thebone surfaces, and osteocytes are present. This bone is histologicallynormal. It is also observed that neovascularization is very marked andthat the cutaneous connective tissue has not proliferated in anuncontrolled manner. In addition, the cutaneous scarring is of anexcellent quality, with no proliferative phenomenon.

-   c) Comparative experiments

The cicatrizing properties of the polysaccharide excreted by the Vibriodiabolicus bacterium were compared with three other polysaccharides,named A, B and C, in table I below.

Polysaccharide A is a high molecular weight (of the order of 1 milliong/mol) fucan of Phaeophyceae.

Polysaccharide B is produced by the strain of Alteromonas macleodiisubsp. fijiensis named ST 716, described by Raguenes et al. in Appliedand Environmental Microbiology, 1996, 62(1), 67–73 and deposited byIFREMER, according to the Treaty of Budapest, on Oct. 17, 1995, with theCNCM (Collection nationale de Cultures de Microorganismes [Nationalcollection of cultures of microorganisms]) held by the PasteurInstitute, 28 rue du Docteur Roux, in Paris, under the number I-1627.The production of his polysaccharide in a purified form is described inthe PCT International publication published under the number WO99/67411.

As described in the PCT International publication published under thenumber WO 99/67411, polysaccharide B consists of glucose, galactose,glucuronic acid, galacturonic acid and pyruvate mannose, these variousconstituents being respectively represented in the molar ratios1/1/1/2/1, and associated in a hexasaccharide repeat unit in which threesaccharide residues form a main chain, the branching point of whichconsists of a galacturonic acid residue. Grafted onto the latter is aside chain which terminates with a mannose residue with a pyruvate atposition 4 and at position 6.

A polysaccharide B consists of the basic hexasaccharide unitcorresponding to the formula (I) below:

Polysaccharide C is produced by precipitation, using 40% (by volume)ethanol, from culture supernatants of the Pseudoalteromonas strain namedHYD 721, as described in the article by H. Rougeaux et al. published inCarbohydrate Research, 1999, 315, 273–285. The basic octasaccharide unitof this polysaccharide corresponds to the formula (II) below:

To assess the cicatrizing activity of the three polysaccharides A, B andC and the polysaccharide excreted by the Vibrio diabolicus bacterium, asurgical protocol identical to that described in point a) above wasused. The results were quantified by comparing the surface of the newlyformed bone relative to that of orifices made initially, this comparisonbeing interpreted in terms of percentage filling of the orifices. TableI below therefore gives the number of animals for which zero (0%),slight (up to 25%), average (up to 50%), extensive (up to 99%) orcomplete (100%) filling of the orifices is observed.

TABLE I Filling of orifices: Total number aver- exten- com- Polymer ofanimals zero slight age sive plete A 3 3 0 0 0 0 B 10 0 2 6 2 0 C 5 4 10 0 0 polysaccharide 8 0 0 0 1 7 excreted by the Vibrio diabolicusspecies

It appears therefore that, among the polysaccharides tested, only thepolysaccharide excreted by the Vibrio diabolicus bacterium allowscomplete repair of orifices formed in the rat calvaria.

-   d) Conclusion

Thus, the polysaccharide excreted by the Vibrio diabolicus bacteriummakes it possible, reproducibly and in approximately two weeks, to fillcritical size bone defects (5 mm in diameter) in rat calvaria. Tests on8 mm cavities and with a 4-week time period also allowed complete repairof a good quality. The critical lesions reconstitute ad-integrum withoutthe uncontrolled effects observed with the use of BMPs. Thepolysaccharide used does not cause any inflammatory reaction and iscompletely resorbed by the surrounding tissues. The polysaccharideexcreted by the Vibrio diabolicus bacterium therefore constitutes amaterial which potentiates bone repair. Its action is due to itsoriginal physicochemical characteristics. It also induces 80% repair ofthe control orifice, over the same period of time.

1. A bone reconstruction biomaterial, comprising a polysaccharideexcreted by the Vibrio diabolicus species having cicatrizing activity,and at least one of an osteoconductive or osteoinductive fillermaterial.
 2. A bone endoprosthesis at least partially coated with apolysaccharide excreted by the Vibrio diabolicus species havingcicatrizing activity.
 3. A method for cicatrizing bone comprising:administering to a subject in need thereof a polysaccharide excreted bythe Vibrio diabolicus species to said bone in an amount effective forcicatrizing the bone.
 4. The method of claim 3, wherein thepolysaccharide is administered in an amount effective for repairing thebone.
 5. The method of claim 3, further comprising coating a boneendoprosthesis with the polysaccharide.
 6. The method of claim 3,further comprising administering an osteoconductive filling material tobone.
 7. The method of claim 3, wherein the polysaccharide is obtainedby precipitation with ethanol from culture supernatants of the Vibriodiabolicus species.
 8. The method of claim 3, wherein the polysaccharidedoes not comprise any neutral polysaccharides, has an acid saccharidecontent of 50% by weight, has an osamine content of 50% by weight, andhas a glucoronic acid/N-acetylgalactosamine/N-acetylglucosaminemonosaccharide molar ratio of 2/1/1.
 9. The method of claim 3, whereinthe polysaccharide is present in a composition at least one of anosteoconductive or osteoinductive filler material.
 10. The methos ofclaim 9, comprising administering a composition comprising anosteoconductive material selected from the group consisting of adeproteinized bone, deceramized bolne, a synthetic calcium phosphate, anatural calcium phophate, biovitroceramic and a bioglass.